Acoustically ejecting a droplet of fluid from a reservoir by an acoustic fluid ejection apparatus

ABSTRACT

The invention provides apparatuses and methods for acoustically ejecting the fluid from a reservoir contained in or disposed on a substrate. The reservoir has a portion adapted to contain a fluid, and an acoustic radiation generator is positioned in acoustic coupling relationship to the reservoir. Acoustic radiation generated by the acoustic radiation generator is transmitted through at least the portion of the reservoir to an analyzer. The analyzer is capable of determining the energy level of the transmitted acoustic radiation and raising the energy level of subsequent poises to a level sufficient to eject fluid droplets from the reservoir. The invention is particularly suited for delivering fluid from a plurality of reservoirs in an accurate and efficient manner.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/956,616, filed Oct. 1, 2004, which is incorporated by reference inits entirety.

TECHNICAL FIELD

The invention relates generally to the use of acoustic energy foracoustically ejecting fluid disposed in a reservoir contained in ordisposed on a substrate. In particular, the invention relates to methodsand apparatuses used in the analysis and adjustment of acoustic energylevels to a level sufficient to provide a droplet-forming pulse. Uniformenergy levels in droplet formation and ejection are more likely toproduce greater uniformity in drop volume and more effective control onpower output of the ejector. With known power levels for dropletejection, it is possible to develop a “signature” for one well or for aplurality of wells disposed in or contained on a well plate. With aplate of known “signature” it is possible to calibrate the ejectoritself to determine its power settings. The invention is particularlysuited for use in conjunction with combinatorial synthetic andanalytical systems that employ biomolecular libraries containing a largenumber of different fluid reservoirs.

BACKGROUND

The discovery of novel and useful materials depends largely on thecapacity to make and characterize new compositions of matter. As aresult, recent research relating to novel materials having usefulbiological, chemical, and/or physical properties has focused on thedevelopment and implementation of new methods and systems forsynthesizing and evaluating potentially useful chemical compounds. Inparticular, high-speed combinational methods have been developed toaddress the general need in the art for systematic, efficient, andeconomical material synthesis techniques as well as methods to analyzeand to screen novel materials for useful properties.

High-speed combinatorial methods often involve the use of arraytechnologies that require accurate dispensing of fluids each having aprecisely knows chemical composition, concentration, stoichiometry,ratio of reagents, and/or volume. Such array technologies may beemployed to carry out various synthetic processes and evaluations. Arraytechnologies may employ large numbers of different fluids to form aplurality of reservoirs that, when arranged appropriately, createcombinatorial libraries. In order to carry out combinatorial techniques,a number of fluid dispensing techniques have been explored, such as pinspotting, pipetting, inkjet printing, and acoustic ejection.

Many of these techniques possess Inherent drawbacks that must beaddressed, however, before the fluid dispensing accuracy and efficiencyrequired for the combinatorial methods can be achieved. For instance, anumber of fluid dispensing systems are constructed using networks oftubing or other fluid-transporting vessels. Tubing, in particular, canentrap air bubbles, and nozzles may become clogged by lodgedparticulates. As a result, system failure may occur and cause spuriousresults. Furthermore, cross-contamination between the reservoirs ofcompound libraries may occur due to inadequate flushing of tubing andpipette tips between fluid transfer events. Cross-contamination caneasily lead to inaccurate and misleading results.

Acoustic ejection provides a number of advantages over other fluiddispensing technologies. In contrast to inkjet devices, nozzleless fluidejection devices are not subject to clogging and their associateddisadvantages, e.g., misdirected fluid or improperly sized droplets.Furthermore, acoustic technology does not require the use of tubing orinvolve invasive mechanical actions, for example, those associated withthe introduction of a pipette tip into a reservoir of fluid.

Acoustic ejection has been described in a number of patents. Forexample, U.S. Pat. No. 4,308,547 to Lovelady et al. describes a liquiddrop emitter that utilizes acoustic principles to eject droplets from abody of liquid onto a moving document to result in the formation ofcharacters or barcodes thereon. A nozzleless inkjet printing apparatusis used such that controlled drops of ink are propelled by an acousticalforce produced by a curved transducer at or below the surface of theink. Similarly, U.S. Ser. No. 09/964,212 describes a device foracoustically ejecting a plurality of fluid droplets toward discretesites on a substrate surface for deposition thereon. The device includesan acoustic radiation generator that may be used to eject fluid dropletsfrom a reservoir, as well as to produce a detection acoustic wave thatis transmitted to the fluid surface of the reservoir to become areflected acoustic wave. Characteristics of the reflected acousticradiation may then be analyzed in order to assess the acoustic energylevel produced by the acoustic radiation generator at the fluid surface.Thus, acoustic ejection may provide an added advantage in that theproper use of acoustic radiation provides feedback relating to theprocess of acoustic ejection itself.

The ability to predetermine the threshold level of droplet productionfor a fluid disposed in a reservoir would enable the user to moreaccurately control droplet size, minimize fluid waste and providesubstantially more effective control of the power output of the acousticenergy generating apparatus used in fluid output from the reservoirs.Because the materials having biological, chemical, and/or physicalproperties useful in combinatorial synthesis can be extremely rareand/or prohibitively expensive, it is desirable to provide effectivecontrols on power output, and consequently drop volume, in their use.

Regardless of the dispensing technique used, however, inventory andmaterials handling limitations generally dictate the capacity ofcombinatorial methods to synthesize and analyze increasing numbers ofsample materials. For instance, during the formatting and dispensingprocesses, microplates that contain a plurality of fluids in individualwells may be thawed, and the fluid in selected wells can then beextracted for use in a combinatorial method. When a pipetting system isemployed during extraction, a minimum loading volume may be required forthe system to function properly. Similarly, other fluid dispensingsystems may also require a certain minimum reservoir volume to functionproperly. Thus, for any fluid dispensing system, it is important toaudit or monitor the reservoir contents to ensure that at least aminimum amount of fluid is provided. Such content monitoring generallyserves to indicate the overall performance of a fluid dispensing system,as well as to maintain the integrity of the combinatorial methods.

An additional feature desirable in fluid monitoring is the ability toevaluate the properties of the microplate itself. Structuralanisotropies at the molecular level, such as variations in the molecularorientation in the polymers used to make the plate, can impact thetransmission of acoustic energy through the plate. Variations inmolecular orientation lead to variations in the reflected acousticenergy from both the plate interface with the reservoir fluid as well asthe reservoir fluid interlace with the atmosphere. Such variations needto be accounted for or they will be attributed erroneously to variationsin composition measurements, fluid height detection and the amount ofenergy reaching the surface.

In addition, during combinatorial synthesis or analysis processes,environmental effects may play a role in altering the reservoircontents. For example, dimethylsulfoxide (DMSO) is a common organicsolvent employed to dissolve or suspend compounds commonly found in druglibraries. DMSO is highly hygroscopic and tends to absorb any ambientwater with which it comes into contact. In turn, the absorption of waterdilutes the concentration the compounds as well as alters the ability ofthe DMSO to suspend the compounds. Furthermore, the absorption of watermay impact the transmission properties of acoustic energy transmittedthrough a DMSO/water mixture and other water-sensitive compounds.

U.S. Pat. No. 5,880,364 to Dam, on the other hand, describes anon-contact ultrasonic system for measuring the volume of liquid in aplurality of containers. An ultrasonic sensor is disposed opposite thetop of the containers. A narrow beam of ultrasonic radiation istransmitted from the sensor to the open top of an opposing container tobe reflected from the air-liquid interface of the container back to thesensor. By using the round trip transit time of the radiation and thedimensions of the containers being measured, the volume of liquid in thecontainer can be calculated. This device cannot be used to analyze waveforms of acoustic energy in fluid in sealed containers. In addition, thedevice lacks precision because air is a poor conductor of acousticenergy. Thus, while this device may provide rough estimate of the volumeof liquid in relatively large containers, it is unsuitable for use inproviding a detailed analysis of the wave forms of acoustic energy influids in reservoirs typically used in combinatorial techniques. Inparticular, this device cannot determine the position of the bottom ofcontainers since substantially all of the emitted acoustic energy isreflected from the liquid surface and does not penetrate to detect thebottom. Small volume reservoirs such as microplates are regular arraysof fluid containers, and the location of the bottoms of the containerscan vary by a significant fraction of the nominal height of a containerdue to distortions in the plate, such as bowing. Thus, detection of onlythe position of the liquid surface leads to significant errors in heightand thus volume estimation in common containers.

Thus, there is a need in the art for improved methods and apparatusesthat are capable of efficiently delivering fluid to a plurality ofreservoirs, a capability that is particularly useful in synthetic andanalytical processes to increase the robustness, efficiency, andeffectiveness of the combinatorial techniques employed therein.

There is a need in the art to determine the energy level of an acousticpulse to a site at the surface of a fluid in a reservoir and the abilityto process the reflected energy of that pulse to be able to raise theamplitude of succeeding pulses to an energy level sufficient to form adroplet, i.e., threshold level. There is a need to analyze the inputenergy level of a pulse having an energy level sufficient to disturb orperturb the surface of the fluid in the reservoir but lower manthreshold level, i.e., a sub-threshold pulse, in order to be able togenerate a subsequent pulse having a sufficient acoustic energy level toform a droplet. There is a need in the art for a method to mapnon-uniformities in the wells contained in a well plate. There is a needin the art to be able to effectively calibrate the power system used togenerate acoustic energy.

SUMMARY OF THE INVENTION

In a first embodiment, the invention provides an improved method foracoustically ejecting a droplet of fluid from a fluid reservoircontained in or located on a substrate. The reservoir containing a fluidis acoustically coupled to an acoustic ejector that produces acousticradiation. The ejector is activated to generate pulses of acousticradiation through the substrate, to a site at or near the surface of thefluid in the reservoir, in a manner effective to eject a droplet fromthe reservoir. In the improved method, prior to acoustically ejecting adroplet of fluid, the wave form of a sub-threshold or perturbation pulseof acoustic radiation to be generated at said site is determined and theamplitude of the perturbation pulse of acoustic radiation generated atthe site is adjusted to an acoustic energy output level sufficient toeject a fluid droplet.

In the method of the first embodiment, the amplitude of the drop formingpulse at a site is determined by acoustically coupling the acousticejector with the reservoir, activating the acoustic ejector to generateand direct a perturbation pulse at the site such that no fluid dropletwill be ejected, and then generating a perturbation interrogation pulseto the fluid surface of the site. The perturbation interrogation pulseis reflected from the fluid surface. The analyzer then detects andprocesses the reflected perturbation interrogation pulse.

The processing of the reflected perturbation interrogation pulse mayinclude analysis of the time domain waveform of the pulse of itsfrequency spectrum. Analysis of the frequency spectrum includes using afrequency domain-based algorithm to identify the difference in frequencyspacing between two minima of an “echo” portion of the reflectedperturbation interrogation pulse. The spacing between two minima is thenused by the analyzer to increase the acoustic energy level of theperturbation pulse by the acoustic ejector to a level sufficient toeject a fluid droplet. In a preferred embodiment of the improved method,the frequency domain-based adaptive algorithm utilizes a Fast FourierTransform (FFT) to characterize the frequency content of theperturbation interrogation pulse response and extract the minima.

In another embodiment, the invention relates to an apparatus foracoustically ejecting a droplet of fluid from a reservoir wherein thereservoir is contained in or disposed on a substrate and a quantity offluid is disposed in the reservoir. The apparatus includes an acousticradiation generator for generating a pulse of acoustic radiation and ameans for acoustically coupling the acoustic radiation generator withthe reservoir.

The acoustic radiation generator can generate a pulse of acousticradiation which is then transmitted through the substrate to a site ator near the surface of the fluid in the reservoir. Such pulse isintended to eject a droplet from the reservoir acoustically coupled tothe acoustic radiation generator.

The apparatus includes an analyzer for determining, prior toacoustically ejecting a droplet of fluid from a reservoir, the energylevel of a perturbation pulse of acoustic radiation to be generated atsaid site. The analyzer then adjusts the amplitude of the perturbationpulse to an acoustic energy output level sufficient to eject a fluiddroplet.

A further embodiment relates to an apparatus for acoustically ejecting adroplet of fluid from each of a plurality of reservoirs wherein suchapparatus includes a plurality of fluid reservoirs contained in orlocated on one or more substrates and a quantity of fluid disposed ineach of the reservoirs.

In the apparatus of this further embodiment the acoustic radiationgenerator is successively acoustically coupled with each reservoir suchthat a pulse of acoustic radiation generated by the acoustic radiategenerator is transmitted through the substrate and into the fluid to asite at or near the surface of the fluid in each reservoir in a mannerintended to eject a droplet from each reservoir.

Prior to acoustically ejecting a droplet of fluid from a reservoir theanalyzer for this embodiment then determines, successively, for each ofthe reservoirs contained in or disposed on the substrate, the wave formof a perturbation pulse of acoustic radiation to be generated at eachsite. The determination of each perturbation pulse wave form is basedtypically on the wave form for a drop forming pulse for desired dropletvolume for the fluid composition in each reservoir. The analyzer thenfellows the perturbation pulse with one or more perturbationinterrogation pulses to adjust the amplitude of succeeding pulses ineach reservoir to an acoustic energy output level sufficient to ejectfluid droplets.

In the apparatus of this further embodiment, the analyzer determines theamplitude necessary for a drop forming pulse at each site byacoustically coupling the acoustic ejector successively with eachreservoir, then activating the acoustic ejector to generate and direct aperturbation pulse at a site in each reservoir. The perturbation pulseis sufficient to disturb the surface of the fluid at the site, but isnot at an acoustic energy level sufficient to allow a fluid droplet tobe ejected from the site. Following the perturbation pulse, the analyzercauses the ejector to generate a perturbation interrogation pulse to thefluid surface of each site. Typically the perturbation interrogationpulse is reflected from the fluid surface. The analyzer then detects andprocesses the reflected perturbation interrogation pulse.

The processing of the reflected perturbation interrogation pulse by theanalyzer includes analyzing echo data provided within such pulse.Analysis of the echo data may include either time-domain orfrequency-domain analysis such as identification of the difference infrequency spacing between two minima of the processed echo data.

In one specific embodiment, the analyzer then uses the spacing betweentwo minima to determine the acoustic energy level necessary for theacoustic ejector to produce a droplet forming pulse. In a preferredembodiment of the apparatus, the frequency domain-based adaptivealgorithm used by the analyzer to process the echo data is an FFT-basedalgorithm.

A further embodiment of the present invention provides a method foracoustically auditing a plurality of fluid reservoirs contained in orlocated on a substrate. An acoustic ejector that produces acousticradiation is acoustically coupled to a first reservoir at a first sitecontaining a fluid.

The ejector is then activated to generate a perturbation pulse ofacoustic radiation through the substrate and into the fluid sufficientto disturb the surface of the fluid at the site but below the acousticenergy level effective to eject a droplet from the site. The fluidvolume in the reservoir is adjusted to conform to a predeterminedperturbation level at the site; optionally, the ejector can be activatedto generate a pulse of acoustic radiation through the substrate and intothe fluid in a manner effective to eject a droplet from the firstreservoir. The method set forth above is then repeated with each of theplurality of fluid reservoirs on the substrate in succession. The volumeadjustments obtained at each site are used to catalogue site to sitevariations on the substrate. Optionally, data associated with the typeof substrate/reservoirs used—for example, a given type of microplate—canbe stored and used when the same plate type is encountered again tofacilitate the droplet forming process. The present invention alsoprovides a method for acoustically auditing a plurality of fluidreservoirs contained in or located on a substrate by providing adeterminable volume fluid having a known composition in each reservoirand adjusting the fluid volume in each reservoir to conform the site toa predetermined ejection threshold level, acoustically coupling a firstreservoir at a first site containing a fluid to an acoustic ejector thatproduces acoustic radiation, then activating the ejector to generate asub threshold pulse of acoustic radiation through the substrate and intothe fluid to the site below the level effective to eject a droplet fromthe first reservoir, and analyzing the sub threshold pulse to determinethe gap to the election threshold. The ejection threshold is thencompared with the predetermined ejection threshold. The procedure isthen repeated with each of the plurality of fluid reservoirs insuccession, then, the difference between ejection threshold and apredetermined ejection threshold at each site is used to catalogue siteto site variations on the substrate.

In another method for acoustically ejecting a droplet of fluid in one ormore fluid reservoirs, the analyzer is operated to analyze acharacteristic of the transmitted, radiation in order to assess thefluid in a selected reservoir. Optionally, the acoustic radiationgenerator could be coupled acoustically successively to each of theremaining reservoirs to permit assessment of the fluid therein.

To determine the volume of fluid in a selected reservoir, we must firstdetermine its composition based on impedance Information based on theratio of the reflected energy amplitude from the fluid/reservoirinterface and the reflected energy amplitude from the fluid surface.This measurement provides the speed of sound. Then the time delaybetween reflected signals from the upper and lower interfaces of theliquid is used to provide the travel time within the fluid. Time (t)multiplied by the speed-of-sound (v_(s)), with the result divided by 2,i.e., (t*v_(s)/2), provides the depth of the fluid in the well. Volumecan then be estimated for a known cavity shape for a well. Factorsinfluencing such volume calculation include the ability to estimate howmuch fluid is in the meniscus. The results of the acoustic analysis maybe stored electronically for later use.

One object of the present invention is to determine the right amount ofenergy for ejection in the droplet-forming application. However thedroplet forming application can also be used for calibration purposes.In a first calibration method using the droplet forming application,reflected energy can be used to calibrate the energy generation anddelivery system. In a second calibration method using thedroplet-forming application, the impact of the energy transmittedthrough the plate can be determined in order to correct for variations(well-to-well) in the micro plate as well as in other micro-platesbelieved to be similar in behavior because they were produced in asimilar fashion (using the same mold, the same molded materials and thesame molding parameters).

Typically, the inventive apparatus includes a single acoustic radiationgenerator and a plurality of removable reservoirs. In addition, theacoustic radiation generator may comprise a component common to theanalyzer, such as a piezoelectric element. Optionally, the acousticgenerator may represent a component of an acoustic ejector, which ejectsdroplets from the reservoirs. In such a case, the apparatus may furthercomprise a means to focus the acoustic radiation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B, collectively referred to as FIG. 1, schematicallyillustrate m simplified cross-sectional view a preferred embodiment ofthe inventive apparatus that allows both the ejection of fluid dropletsfrom a plurality of reservoirs and the adjustment of the amplitude ofthe pulse of acoustic radiation generated at each individual site to anacoustic energy output level sufficient to eject a fluid droplet. Asdepicted, the apparatus comprises first and second reservoirs, acombined acoustic analyzer and ejector, and an ejector positioningmeans. FIG. 1A shows the acoustic ejector acoustically coupled to thefirst reservoir; the ejector is activated in order to eject a droplet offluid from within the first reservoir toward a site on a substratesurface to form an array. FIG. 1B shows the acoustic ejectoracoustically coupled to a second reservoir.

FIG. 2 schematically illustrates in simplified cross-sectional view anembodiment of the inventive apparatus designed to process informationobtained from the reflective acoustic energy obtained from a wave formgenerated by the ejector.

FIGS. 3A-3C, collectively referred to as FIG. 3, schematicallyillustrate a rectilinear array of reservoirs in the form of a well platehaving three rows and two columns of wells each having a lowheight-to-diameter ratio. FIG. 3A illustrates a well plate in top view.FIG. 3B illustrates the well plate in cross-sectional view along dottedline A. FIG. 3C illustrates the well plate in bottom view.

FIGS. 4A-4C, collectively referred to as FIG. 4, are a series of imagesof the perturbed surface of a fluid in a reservoir. FIG. 4A showsright-hand and left-hand images of DMSO/water mixtures having DMSOconcentrations of 70% and 90%, respectively, for tone burst excitation 1dB below threshold. FIG. 4B shows right-hand and left-hand images ofDMSO/water mixtures having DMSO concentrations of 70% and 90%,respectively, for tone burst excitation 0.5 dB below threshold. FIG. 4Cshows right-hand and left-hand images of DMSO/water mixtures having DMSOconcentrations of 70% and 90%, respectively, for tone burst excitationat ejection threshold.

FIG. 5 is a chart showing a chart of the spacing between two minima forthe tone burst inputs and the DMSO concentrations associated with FIGS.4A-4C.

FIG. 6 is a flow chart of the algorithm for the spacing between twominima of the present invention.

FIGS. 7A-7C, collectively referred to as FIG. 7, illustrate the waveforms processed by the analyzer using the algorithm of FIG. 6. FIG. 7Ashows a perturbation interrogation pulse response waveform. FIG. 7Bshows a perturbation interrogation pulse “echo” waveform. FIG. 7C showsa Fast Fourier Transform (FFT) obtained from the processing of theperturbation interrogation pulse “echo” waveform of FIG. 7B.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific fluids,biomolecules, or apparatus structures, as such may vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a reservoir” includes a plurality of reservoirs, referenceto “a fluid” includes a plurality of fluids, reference to “abiomolecule” includes a combination of biomolecules, and the like.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set forthbelow.

The terms “acoustic coupling” and “acoustically coupled” as used hereinrefer to a state wherein an object is placed in direct or indirectcontact with another object so as to allow acoustic radiation to betransferred between the objects without substantial loss of acousticenergy. When two entities are indirectly acoustically coupled, an“acoustic coupling medium” is needed to provide an intermediary throughwhich acoustic radiation may be transmitted. Thus, an ejector may beacoustically coupled to a fluid, such as by immersing the ejector in thefluid, or by interposing an acoustic coupling medium between the ejectorand the fluid, in order to transfer acoustic radiation generated by theejector through the acoustic coupling medium and into the fluid. Thegenerator and the fluid can be brought into an acoustic couplingrelationship through either the motion of the generator, the reservoircontaining the fluid or both.

The term “fluid” as used herein refers to matter that is nonsolid, or atleast partially gaseous and/or liquid, but not entirely gaseous. A fluidmay contain a solid that is minimally, partially, or fully solvated,dispersed, or suspended. Examples of fluids include, without limitation,aqueous liquids (including water per se and salt water) and nonaqueousliquids such as organic solvents and the like. As used herein, the term“fluid” is not synonymous with the term “ink” in that an ink mustcontain a colorant and may not be gaseous.

The terms “focusing means” and “acoustic focusing means” refer to ameans for causing acoustic waves to converge at a focal point, either bya device separate from the acoustic energy source that acts like anoptical lens, or by the spatial arrangement of acoustic energy sourcesto effect convergence of acoustic energy at a focal point byconstructive and destructive interference. A focusing means may be assimple as a solid member having a curved surface, or it may includecomplex structures such as those found in Fresnel lenses, which employdiffraction in order to direct acoustic radiation. Suitable focusingmeans also include phased array methods as are known in the art anddescribed, for example, in U.S. Pat. No. 5,798,779 to Nakayasu et al.and Amemiya et al. (1997) Proceedings of the 1997 IS&T NIP13International Conference on Digital Printing Technologies, pp. 698-702.

The terms “library” and “combinatorial library” are used interchangeablyherein to refer to a plurality of chemical or biological moietiesarranged in a pattern or an array such that the moieties areindividually addressable. In some instances, the plurality of chemicalor biological moieties is present on the surface of a substrate, and inother instances, the plurality of moieties represents the fluid in aplurality of reservoirs. Preferably, but not necessarily, each moiety isdifferent from each of the other moieties. The moieties may be, forexample, peptidic molecules and/or oligonucleotides.

The term “low frequency preamble” refers to a precursor segment of theperturbation interrogation pulse echo in the time domain. If present, itis desirable to eliminate the low frequency preamble from the frequencycontent analysis of the perturbation interrogation pulse echo. This canbe accomplished by including only the portion of the perturbationinterrogation pulse echo which matches the frequency content of theperturbation interrogation pulse. The FFT of the time domain signal ofthe perturbation interrogation pulse echo with the low frequencypreamble removed is then used for determination of the spacing betweentwo minima. The low frequency preamble deletion from the time seriesinput to the FFT improves robustness of the signal processing bypreventing a shift in the location of the lowest frequency minimum.

The term “moiety” refers to any particular composition of matter, e.g.,a molecular fragment, an intact molecule (including a monomericmolecule, an oligomeric molecule, and a polymer), or a mixture ofmaterials (for example, an alloy or a laminate).

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

The term “perturbation pulse” refers to the acoustic energy delivered toa site in the fluid to create a response in the surface. Typically, thisenergy has a similar duration and a similar frequency spectrum as thedrop forming pulse, but this pulse has lower amplitude than the dropletforming pulse. In other words, this pulse is designed to deliver what issubstantially a lower amplitude (scaled-down) version of the dropletforming pulse to the site in order to evoke the same energy transmissioncharacteristics as the droplet forming pulse. Acoustic transduction bythe acoustic generator and acoustic energy transmission from theacoustic generator to the site is not uniform for all frequencies, andhence, it is desirable to have the same frequency content for theperturbation pulse as is in the droplet forming pulse. Also, the time atwhich energy of a given frequency arrives at the site will influence thedynamics of the perturbation. Hence, it is desirable for the relativetime of the arrival of energy of various frequencies to be similar inthe perturbation and droplet forming pulses. The term “pulse” and theterm “tone burst” both refer to waveforms, with the only differenceoften being the number of cycles in the waveform, and may be usedinterchangeably throughout the subject patent application.

The term “perturbation interrogation pulse” refers to the acousticenergy delivered to the site in the fluid to determine the response ofthe fluid to the perturbation pulse. The pulse is generated at a timeinterval following the perturbation pulse that enables the surface torespond to the perturbation pulse. The energy reflected from theperturbed surface is referred to herein as the perturbationinterrogation pulse response.

The term “perturbation interrogation pulse echo” refers to that portionof the perturbation interrogation pulse response that is used by theanalyzer to determine the spacing between two minima used to adjust theamplitude of a perturbation pulse to a droplet forming level. Theperturbation interrogation pulse echo is relatively short in duration(about 1 MHz) and occurs about 550 to 575 MHz after the beginning of theperturbation interrogation pulse response.

The term “ranging interrogation pulse” refers to the acoustic pulse usedto find the depth of the liquid in a well in order to adjust thetransducer position such that the site is located near the fluid surfacefor drop generation.

The term “reservoir” as used herein refers to a receptacle or chamberfor containing a fluid. In some instances, a fluid contained in areservoir necessarily will have a free surface, e.g., a surface thatallows acoustic radiation to be reflected there from or a surface fromwhich a droplet may be acoustically ejected. A reservoir may also be alocus on a substrate surface within which a fluid is constrained.

The term “site” or “energy site” refers to the location in the fluidthat will receive acoustic energy. This can be for different purposessuch as for perturbing the surface, interrogating the perturbation ofthe surface or forming a drop. The site is preferably at or near thesurface of a fluid in a reservoir.

The term “substrate” as used herein refers to any material having asurface which provides means for containing one or more fluid volumes.The substrate may be constructed in any of a number of forms including,for example, wafers, slides, well plates, or membranes. In addition, thesubstrate may be porous or nonporous as required for containment of aparticular fluid volume. The means for containing fluid volumes areoften reservoirs of wells. Suitable substrate materials include, but arenot limited to, supports that are typically used for solid phasechemical synthesis, such as polymeric materials (e.g., polystyrene,polyvinyl acetate, polyvinyl chloride, polyvinyl pyrrolidone,polyacrylonitrile, polyacrylamide, polymethyl methacrylate,polytetrafluoroethylene, polyethylene, polypropylene, polyvinylidenefluoride, polycarbonate, and divinylbenzene styrene-based polymers),agarose (e.g., Sepharose®), dextran (e.g., Sephadex®), cellulosicpolymers and other polysaccharides, silica and silica-based materials,glass (particularly controlled pore glass, or “CPG”) and functionalizedglasses, ceramics, such substrates treated with surface coatings, e.g.,with microporous polymers (particularly cellulosic polymers such asnitrocellulose), microporous metallic compounds (particularlymicroporous aluminum) antibody-binding proteins (available from PierceChemical Co., Rockford Ill.), bisphenol A polycarbonate, or the like.Additional, information relating to the term “substrate” can be found inU.S. Ser. No. 09/964,212.

The invention accordingly relates to apparatuses and methods foracoustically ejecting fluid from a fluid reservoir. The inventive methodprovides an improved method for acoustically ejecting a droplet of fluidfrom a fluid reservoir contained in or located on a substrate. Thereservoir containing a fluid is acoustically coupled to an acousticejector that produces acoustic radiation. The ejector is activated togenerate pulses of acoustic radiation through the substrate, to a siteat or near the surface of the fluid in the reservoir, in a mannereffective to eject a droplet from the reservoir. In the improved method,prior to acoustically ejecting a droplet of fluid, the wave form of aperturbation pulse of acoustic radiation to be generated at said site isdetermined and the amplitude of the perturbation pulse of acousticradiation generated at the site is adjusted to an acoustic energy outputlevel sufficient to eject a fluid droplet.

In the method of the first embodiment, the amplitude of the drop formingpulse at a site is determined by acoustically coupling the acousticejector with the reservoir, activating the acoustic ejector to generateand direct a perturbation pulse at the site such that no fluid dropletwill be ejected, and then generating a perturbation interrogation pulseat the fluid surface of the site. The perturbation interrogation pulseis reflected from the fluid surface. The analyzer then detects andextracts the perturbation interrogation pulse echo from the perturbationinterrogation pulse response for further processing.

The processing of the reflected perturbation interrogation pulseincludes analyzing its frequency spectrum. Analysis of the frequencyspectrum includes using a frequency domain-based algorithm to identifythe difference in frequency spacing between two minima of the echoportion of the reflected perturbation interrogation pulse. Optionally,the perturbation interrogation pulse response or the perturbationinterrogation pulse echo can be processed to remove spurious noise orrestrict the time domain signal to improve robustness of the analysis ofthe resulting FFT. The spacing between two minima is then used by theanalyzer to increase the acoustic energy level of the perturbation pulseby the acoustic ejector to a level sufficient to eject a fluid droplet.In a preferred embodiment of the improved method, the frequencydomain-based adaptive algorithm is a Fast Fourier Transform (FFT)-basedalgorithm.

The inventive apparatus includes a reservoir adapted to contain a fluid,and an acoustic radiation generator for generating acoustic radiation.The inventive apparatus also includes a means for bringing the reservoirand the acoustic radiation generator into an acoustically coupledrelationship such that the acoustic radiation generated by the acousticradiation generator is transmitted through the reservoir. Establishmentof acoustic coupling between the reservoir and the acoustic radiationgenerator may involve either the motion of the reservoir, the acousticgenerator or both. An analyzer for analyzing a pulse of acousticradiation is positioned to receive pulses of acoustic radiationreflected from a site in the fluid in the reservoir. Typically, thepulses to be analyzed are received by the same apparatus that was usedto generate the acoustic radiation as these apparatuses are composed ofpiezoelectric materials and are capable of converting electromagneticenergy to acoustic energy as well as converting acoustic energy backinto electromagnetic energy.

The apparatus may also be constructed for use with a plurality ofreservoirs wherein the reservoirs are an integrated or permanentlyattached component of the apparatus. However, to provide modularity andinterchangeability of components, it is preferred that apparatus beconstructed with removable reservoirs, and that it can operate with aplurality of these removable reservoirs. Generally, the reservoirs arearranged in a pattern or an array to provide each reservoir withindividual systematic addressability. In addition, while each of thereservoirs may be provided as a discrete or stand-alone item, incircumstances that require a large number of reservoirs, it is preferredthat the reservoirs are attached to each other or represent integratedportions of a single reservoir unit.

For example, the reservoirs may represent individual wells in a wellplate. Many well plates suitable for use with the apparatus arecommercially available and may contain, for example, 96, 384, 1536, or3456 wells per well plate. Manufactures of suitable well plates for usein the employed apparatus include Corning, Inc. (Corning, N.Y.) andGreiner America, Inc. (Lake Mary, Fla.). However, the availability ofsuch commercially available well plates does not preclude themanufacture and use of custom-made well plates containing at least about10,000 wells, or as many as 100,000 to 500,000 wells, or more.

Furthermore, the material used in the construction of reservoirs must becompatible with the fluids contained therein. Thus, if it is intendedthat the reservoirs or wells contain an organic solvent such asacetonitrile, polymers that dissolve or swell in acetonitrile would beunsuitable for use in forming the reservoirs or well plates. Similarly,reservoirs or wells intended to contain DMSO must be compatible withDMSO. For water-based fluids, a number of materials are suitable for theconstruction of reservoirs and include, but are not limited to, ceramicssuch as silicon oxide and aluminum oxide, metals such as stainless steeland platinum, and polymers such as polyester, polypropylene, cyclicolefin copolymer, polystyrene, polycarbonate andpolytetrafluoroethylene. For fluids that are photosensitive, thereservoirs may be constructed from an optically opaque material that hassufficient acoustic transparency for substantially unimpairedfunctioning of the apparatus.

In addition, to reduce the amount of movement and time needed to alignthe acoustic radiation generator with each reservoir or reservoir wellduring operation, discussed infra, it is preferable that the center ofeach reservoir be located not more than about 1 centimeter, preferablynot more than about 1 millimeter, and optimally not more than about 0.5millimeter, from a neighboring reservoir center. These dimensions tendto limit the she of the reservoirs to a maximum volume. The reservoirsare constructed to contain typically no more than about 1 mL, preferablyno more than about 1 μL, and optimally no more than about 1 nL, offluid. To facilitate handling of multiple reservoirs, it is alsopreferred that the reservoirs be substantially acousticallyindistinguishable.

Generally, a single acoustic radiation generator is employed, though aplurality of acoustic radiation generators may be employed as well. Allacoustic radiation generators employ a vibrational element or transducerto generate acoustic radiation. Often, a piezoelectric element isemployed to convert electrical energy into mechanical energy associatedwith acoustic radiation. When a single acoustic radiation generator isemployed, the positioning means should allow for the acoustic radiationgenerator to move from one reservoir to another quickly and in acontrolled manner, thereby allowing fast and controlled scanning of thefluid in the reservoirs.

In order to ensure optimal performance, it is important to keep in mindthat there are two basic kinds of motion: pulse and continuous. Pulsemotion involves the discrete steps of coupling an acoustic radiationgenerator to a reservoir, keeping it stationary while it emits acousticenergy, and then coupling the generator to another reservoir, using ahigh performance positioning means with such a method allows repeatableand controlled acoustic coupling at each reservoir in less than 0.1second. Typically, the pulse width is very short and may enable over 10Hz reservoir transitions and even over 1000 Hz reservoir transitions. Acontinuous motion design, on the other hand, moves the acousticradiation generator and/or the reservoirs continuously, although not atthe same speed.

In some instances, the analyzer is positioned in fixed alignment withrespect to the acoustic radiation generator. In other instances,however, a means similar to that described above is provided foraltering the relative positions of the analyzer with respect to thereservoirs. The relative position of the analyzer and the acousticradiation generator depends on the particular configuration of theapparatus. In some instances, the apparatus may be configured to operatein transmissive mode, such that the generated radiation is transmittedthrough the entirety of the reservoir from which a droplet of fluid isto be ejected. In such a case, the reservoir may be interposed betweenthe acoustic radiation generator and an acoustic analyzer. As anotheroption, the apparatus may be configured to operate in a reflective mode,such that the acoustic radiation is transmitted only through a portionof the reservoir from which a droplet of fluid is to be ejected. In sucha case, the analyzer may be positioned in a manner appropriate for thisconfiguration, e.g., in order to receive reflected acoustic radiation.

In any case, the acoustic radiation generator is positioned such thatgenerated acoustic radiation is transmitted through each reservoir andthe fluid in the reservoir to be reflected from a site at or near thesurface of the fluid for optimal performance. For example, as fluidsordinarily flow to the bottom of containers or are driven there bycentrifugation, the acoustic radiation generator should be positionedsuch that generated acoustic radiation is transmitted through the bottomof a reservoir.

In a preferred configuration, as discussed in detail below, the analyzeris positioned to receive acoustic radiation reflected from a site at ornear a free surface of a fluid contained in each reservoir. In such aconfiguration, the acoustic radiation generator may comprise a componentcommon to the analyzer. The component common to the acoustic radiationgenerator and the analyzer may be a vibrational element that convertsone form of energy into another, e.g., a piezoelectric element thatconverts acoustic/mechanical energy to electrical energy.

The analyzer may be constructed to perform a number of functions. Forexample, the analyzer may be adapted to analyze a pulse of acousticradiation to determine the waveform of a pulse of drop forming acousticradiation to adjust the amplitude of such a pulse to an acoustic energyoutput level sufficient to eject a fluid droplet. In addition, or in thealterative, the analyzer may be adapted to analyze acoustic radiation todetermine a volume of fluid in each reservoir for plate auditingpurposes. Alternatively, a well plate of known properties, comprising aseries of wells each containing a fluid of known volume and knowncomposition could be used to calibrate the power system generatingacoustic energy. Fluid properties that impact pulse amplitude include,but are not limited to, viscosity, surface tension and composition,including solid content, and impurity content.

Thus, the invention also provides a method for acoustically ejecting adroplet of fluid from each of a plurality of reservoirs. The methodinvolves providing a plurality of reservoirs, each reservoir adapted tocontain a fluid, and positioning an acoustic radiation generator and aselected reservoir in acoustic coupling relationship. Once positioned,the acoustic radiation generator is actuated to generate pulses ofacoustic radiation that are transmitted through a selected reservoir andthe fluid therein to a site at or near the surface of the fluid. Thepulses of acoustic energy are then reflected from the site to ananalyzer. The analyzer then processes a poise of reflected acousticradiation to adjust the amplitude of the pulse of acoustic radiationgenerated at each individual site on the substrate to an acoustic energyoutput level sufficient to eject a fluid droplet. In a preferredembodiment of the present invention, the acoustic radiation generator issuccessively acoustically coupled the remaining reservoirs to enable theanalyzer to adjust the amplitude of the pulses of acoustic radiation inthe fluid in the remaining reservoirs to a droplet forming level aswell.

As discussed above, the reservoirs may be constructed to reduce theamount of movement and time needed to couple each of the reservoirs withthe acoustic radiation generator during operation. As a general matterof convenience and efficiency. It is desirable to analyze an entirelibrary of different moieties in a relatively short amount of time,e.g., about one minute. Thus, the inventive method typically allows forthe analysis of the acoustic energy delivered to each site in the fluidin each of the reservoirs at a time between a first and a secondreservoir of about two seconds. Faster analysis times between reservoirsof 0.25 seconds, 0.02 seconds and 0.005 seconds are achievable withpresent day technology as well. Thus, the invention can be operated toanalyze acoustic energy at each site in the fluid in each well of most(if not all) well plates that are currently commercially available.Proper implementation of the inventive method should yield a reservoiranalysis time between reservoirs of 0.001 seconds. Current commerciallyavailable positioning technology allows the acoustic radiation generatorto be acoustically coupled with one reservoir after another, withrepeatable and controlled acoustic coupling at each reservoir, in lessthan about 0.1 second for high performance positioning means and in lessthan about 1 second for ordinary positioning means. A custom designedsystem will allow repeatable and controlled acoustic coupling of theacoustic radiation generator to one reservoir after another within lessthan about 0.001 second.

By analyzing a pulse of acoustic radiation that has been transmitted toa site of the fluid in a selected reservoir, one may accuratelydetermine the amplitude of the pulse in the fluid in the selectedreservoir.

Acoustic ejection as described above may be employed to improve fluiddispensing from each of a plurality of reservoirs adapted to contain afluid. Thus, another embodiment of the invention relates to an apparatusfor ejecting droplets of fluid from each of a plurality of reservoirsadapted to contain a fluid. This apparatus may include any of a numberof known techniques for ejecting droplets of fluid from a reservoirinvolving contact-based fluid dispensing, e.g., pin spotting, pipetting,and inkjet printing, or non-contact based fluid dispensing, e.g.,acoustic ejection. However, the inventive apparatus represents a noveland nonobvious improvement over the fluid dispensing apparatuses knownin the art since it provides for enhanced accuracy and precision influid dispensing through the use of a means for acoustically ejectingdroplets of fluid from a reservoir. The means for acoustically assessingthe fluid in the reservoirs is similar to the previously describedapparatus for ejecting droplets of fluid from each of a plurality offluid reservoirs in that it also comprises an acoustic radiationgenerator for generating acoustic radiation and an analyzer foranalyzing a waveform of acoustic radiation. A means for acousticallycoupling the acoustic radiation generator with each reservoir is used toensure that acoustic radiation generated by the acoustic radiationgenerator is transmitted through each reservoir and the fluid therein toa site at or near the surface of such fluid. Furthermore, the analyzeris coupled to receive the reflected waveform of the transmitted acousticradiation.

As discussed above, acoustic ejection provides a number of advantagesover other fluid dispensing technologies. In addition, compatibleacoustic ejection technology described in U.S. Ser. No. 09/964,212involves an ejector comprising an acoustic radiation generator forgenerating acoustic radiation and a focusing means for focusing theacoustic radiation generated at a focal point within and sufficientlynear the fluid surface in each of a plurality of reservoirs to result inthe ejection of droplets there from. Optionally, a focusing means istypically provided for focusing the acoustic radiation generated by theacoustic generator. In the present invention, any of a variety offocusing means may be employed in conjunction with the acousticgenerator in order to eject droplets from a reservoir through the use offocused acoustic radiation. For example, one or more curved surfaces maybe used to direct acoustic radiation to a focal point near a fluidsurface. One such technique is described in U.S. Pat. No. 4,308,547 toLovelady et al. Focusing means with a curved surface have beenincorporated into the construction of commercially available acoustictransducers such as those manufactured by Panametrics Inc. (Waltham,Mass.). In addition, Fresnel lenses are known in the art for directingacoustic energy at a predetermined focal distance from an object plane.See, e.g., U.S. Pat. No. 5,041,849 to Quate et al. Fresnel lenses mayhave a radial phase profile that diffracts a substantial portion ofacoustic energy into a predetermined diffraction order at diffractionangles that vary radially with respect to the lens. The diffractionangles should be selected to focus the acoustic energy within thediffraction order on a desired object plane. Optimally, the apparatus isadapted to eject fluid from a reservoir according to the results ofacoustic analysis performed by the analyzer.

The apparatus may also provide certain performance-enhancingfunctionalities. For example, the apparatus may include a means forcontrolling the temperature of one or more of the reservoirs. Suchtemperature controlling means may be employed in the inventive apparatusto improve the accuracy of measurement and may be employed regardless ofwhether the apparatus includes a fluid dispensing functionality. In thecase of aqueous fluids, the temperature controlling means should havethe capacity to maintain the reservoirs at a temperature above about 0°C. In addition, the temperature controlling means may be adapted tolower the temperature in the reservoirs. Such temperature lowering maybe required because repeated application of acoustic energy to areservoir of fluid may result in heating of the fluid. Such heating canresult in unwanted changes in fluid properties such as viscosity,surface tension and density. Design and construction of such temperaturecontrolling means are known to one of ordinary skill in the art and maycomprise, e.g., components such a heating element, a cooling element, ora combination thereof. For many biomolecular applications, reservoirs offluids are stored frozen and thawed for use. During use, it is generallydesired that the fluid containing the biomolecule be kept at a constanttemperature, with deviations of no more than about 1° C. or 2° C. therefrom. In addition, for a biomolecular fluid that is particularly heatsensitive, it is preferred that the fluid be kept at a temperature thatdoes not exceed about 10° C. above the melting point of the fluid,preferably at a temperature that does not exceed about 5° C. above themelting point of the fluid. Thus, for example, when thebiomolecule-containing fluid is aqueous, it may be optimal to keep thefluid at about 4° C. during ejection.

Moreover, the apparatus may be adapted to dispense fluids of virtuallyany type and amount desired. The fluid may be aqueous and/or nonaqueous.Examples of fluids include, but are not limited to, aqueous fluidsincluding water per se and water-solvated ionic and non-ionic solutions,organic solvents, lipidic liquids, suspensions of immiscible fluids, andsuspensions or slurries of solids in liquids. Because the invention isreadily adapted for use with high temperatures, fluids such as liquidmetals, ceramic materials, and glasses may be used; see, e.g.,co-pending patent application U.S. Ser. No. 09/669/194 (“Method andApparatus for Generating Droplets of Immiscible Fluids”), inventorsEllson and Mutz, filed on Sep. 25, 2000, and assigned to Picoliter, Inc.(Mountain View, Calif.). Furthermore, because of the precision that ispossible using the inventive technology, the apparatus may be used toeject droplets from a reservoir adapted to contain no more than about100 nanoliters of fluid, preferably no more than 10 nanoliters of fluid.In certain cases, the ejector may be adapted to eject a droplet from areservoir adapted to contain about 1 to about 100 nanoliters of fluid.This is particularly useful when the fluid to be ejected contains rareor expensive biomolecules, wherein it may be desirable to eject dropletshaving a volume of about 1 picoilter or less, e.g., having a volume inthe range of about 0.025 pL to about 1 pL.

Thus, another embodiment of the invention relates to a method fordispensing fluid from one or more reservoirs. Once an acoustic radiationgenerator is positioned, in acoustic coupling relation to a reservoirselected from a plurality of reservoirs, acoustic radiation generated bythe acoustic radiation generator may be transmitted through the selectedreservoir and the fluid therein to a site at or near the surface of suchfluid. The reflected waveform of acoustic radiation is then analyzed inorder to adjust the amplitude of the pulse of acoustic energy in thefluid in the reservoir, and droplets of fluid are dispensed from theselected reservoir according to the adjustment. Typically, the dropletsof fluid are dispensed through acoustic ejection, though the inventivemethod may employ contact-based fluid dispensing either as analternative to or as a supplement to noncontact-based fluid dispensing.Also, while the analysis of acoustic energy is conducted in thereflective mode, it should be apparent to one skilled in the art thatsuch analysis is not limited thereto, but could be easily conducted inthe transmissive mode along the lines suggested in FIG. 2 and discussedinfra. Optionally, the above described processes may be repeated foradditional reservoirs.

As discussed above, fluid may be dispensed from a reservoir by acousticejection. This allows an operator to fine tune the dispensing accordingto the condition of the fluid in the reservoir.

FIG. 1 illustrates a preferred embodiment of the inventive apparatus insimplified cross-sectional view. In this embodiment, the inventiveapparatus allows for acoustic ejection of fluid droplets from aplurality of reservoirs. The inventive apparatus is shown in operationto form a biomolecular array hound to a substrate. As with all figuresreferenced herein, in which like parts are referenced by like numerals.FIG. 1 is not to scale, and certain dimensions may be exaggerated forclarity of presentation. The apparatus 11 includes a plurality ofreservoirs, i.e., at least two reservoirs, with a first reservoirindicated at 13 and a second reservoir indicated at 15. Each is adaptedto contain a fluid having a fluid surface. As shown, the first reservoir13 contains a first fluid 14 and the second reservoir 15 contains asecond fluid 16. Fluids 14 and 16 each have a fluid surface respectivelyindicated at 17 and 19. Fluids 14 and 16 may be the same or different.As shown, the reservoirs are of substantially identical construction soas to be substantially acoustically indistinguishable, but identicalconstruction is not a requirement. The reservoirs are shown as separateremovable components but may, as discussed above, be fixed within aplate 43 or other substrate. For example, the plurality of reservoirsmay comprise individual wells in a well plate, optimally although notnecessarily arranged in an array. Each of the reservoirs 13 and 15 ispreferably axially symmetric as shown, having vertical walls 21 and 23extending upward from circular reservoir bases 25 and 27 and terminatingat openings 29 and 31, respectively, although other reservoir shapes maybe used. The material and thickness of each reservoir base should besuch that acoustic radiation may be transmitted there through and intothe fluid contained within the reservoirs.

The apparatus also includes an acoustic ejector 33 comprised of anacoustic radiation generator 35 for generating acoustic radiation and afocusing means 37 for focusing the acoustic radiation at a focal pointwithin the fluid from which a droplet is to be ejected, near the fluidsurface. The acoustic radiation generator contains a transducer 36,e.g., a piezoelectric element, commonly shared by an analyzer. As shown,a combination unit 38 is provided that both serves as a controller and acomponent of an analyzer. Operating as a controller, the combinationunit 38 provides the piezoelectric element 36 with electrical energythat is converted into mechanical and acoustic energy. Operating as acomponent of an analyzer, the combination unit receives and analyzeselectrical signals from the transducer. The electrical signals areproduced as a result of the absorption and conversion of mechanical andacoustic energy by the transducer.

As shown in FIG. 1, the focusing means 37 may comprise a single solidpiece having a concave surface 39 for focusing acoustic radiation, butthe foe-using means may be constructed in other ways as discussed below.The acoustic ejector 33 is thus adapted to generate and focus acousticradiation so as to eject a droplet of fluid from each of the fluidsurfaces 17 and 19 when acoustically coupled to reservoirs 13 and 15,and thus to fluids 14 and 16, respectively. The acoustic radiationgenerator 35 and the focusing means 37 may function as a single unitcontrolled by a single controller, or they may be independentlycontrolled, depending on the desired performance of the apparatus.Typically, single ejector designs are preferred over multiple ejectordesigns because accuracy of droplet placement and consistency in dropletsize and velocity are more easily achieved with a single ejector.

There are also a number of ways to acoustically couple the ejector 33 toeach individual reservoir and thus to the fluid therein. One suchapproach is through direct contact as is described, for example, in U.S.Pat. No. 4,308,547 to Lovelady et al., wherein a focusing meansconstructed from a hemispherical crystal having segmented electrodes issubmerged in a liquid to be ejected. The aforementioned patent furtherdiscloses that the focusing means may be positioned at or below thesurface of the liquid. However, this approach for acoustically couplingthe focusing means to a fluid is undesirable when the ejector is used toeject different fluids in a plurality of containers or reservoirs, asrepeated cleaning of the focusing means would be required in order toavoid cross-contamination. The cleaning process would necessarilylengthen the transition time between each droplet ejection event. Inaddition, in such a method, fluid would adhere to the ejector as it isremoved from each container, wasting material that may be costly orrare.

Thus, a preferred approach would be to acoustically couple the ejectorto the reservoirs and reservoir fluids without contacting any portion,of the ejector, e.g., the focusing means, with any of the fluids to beejected. To this end, the present invention provides an ejectorpositioning means for positioning the ejector in controlled andrepeatable acoustic coupling with each of the fluids in the reservoirsto eject droplets there from without submerging the ejector therein.This typically involves direct or indirect contact between the ejectorand the external surface of each reservoir. When direct contact is usedin order to acoustically couple the ejector to each reservoir, it ispreferred that the direct contact is wholly conformal to ensureefficient acoustic energy transfer. That is, the ejector and thereservoir should have corresponding surfaces adapted for mating contact.Thus, if acoustic coupling is achieved between the ejector and reservoirthrough the focusing means, it is desirable for the reservoir to have anoutside surface that corresponds to the surface profile of the focusingmeans. Without conformal contact, efficiency and accuracy of acousticenergy transfer may be compromised. In addition, since many focusingmeans have a curved surface, the direct contact approach may necessitatethe use of reservoirs having a specially formed inverse surface.

Optimally, acoustic coupling is achieved between the ejector and each ofthe reservoirs through indirect contact, as illustrated in FIG. 1A. Inthis figure, an acoustic coupling medium 41 is placed between theejector 33 and the base 25 of reservoir 13, with the ejector andreservoir located at a predetermined distance from each other. Theacoustic coupling medium may be an acoustic coupling fluid, preferablyan acoustically homogeneous material in conformal contact with both theacoustic focusing means 37 and each reservoir. In addition, it isimportant to ensure that the fluid medium is substantially free ofmaterial having different acoustic properties than the fluid mediumitself. Furthermore, it is preferred that the acoustic coupling mediumis comprised of a material having acoustic properties that facilitatethe transmission of acoustic radiation without significant attenuationin acoustic pressure and intensity. Also, the acoustic impedance of thecoupling medium should facilitate the transfer of energy from thecoupling medium into the container. As shown, the first reservoir 13 isacoustically coupled to the acoustic focusing means 37, such that anacoustic wave is generated by the acoustic radiation generator anddirected by the focusing means 37 into the acoustic coupling medium 41,which then transmits the acoustic radiation into the reservoir 13.

In operation, reservoirs 13 and 15 are each filled with first and secondfluids 14 and 16, respectively, as shown in FIG. 1. The acoustic ejector33 is positioned by means of plate or ejector positioning means 43,shown below reservoir 13, in order to achieve acoustic coupling betweenthe ejector and the reservoir through acoustic coupling medium 41.Positioning of reservoirs 13 and 15 could be accomplished by a reservoirpositioning means (not shown) disposed between the reservoirs 13 or 15and the plate 43. Once the ejector, the reservoir, and the substrate arein proper alignment, the acoustic radiation generator 35 is activated toproduce acoustic radiation that is directed toward a free fluid surface17 of the first reservoir. The acoustic radiation will then travel in agenerally upward direction toward the tree fluid surface 17. Thewaveform of the reflected acoustic radiation will differ based on thelevel and duration of acoustic energy delivered to the site of the fluidin the reservoir. For example, reflection will occur when there is achange in the acoustic property of the medium through which the acousticradiation is transmitted. A portion of the acoustic radiation travelingupward will be reflected from the reservoir bases 35 and 27 as well asthe free surfaces 17 and 19 of the fluids contained in the reservoirs 13and 15.

As discussed above, acoustic radiation may be employed for use as ananalytical tool as well as to eject droplets from a reservoir. In ananalytical mode, the acoustic radiation generator is typically activatedso as to generate low energy acoustic radiation that has insufficientacoustic energy to eject a droplet from the fluid surface. This istypically done by using an extremely short pulse (on the order of tensof nanoseconds) relative to that required for droplet ejection (on theorder of microseconds). This is the type of pulse used for ranging. Itis neither a perturbation pulse nor is it a perturbation interrogationpulse.

By determining the time it takes for the acoustic radiation to bereflected by the fluid surface back to the acoustic radiation generator,and then correlating that time with the speed of sound in the fluid,that distance, and thus the fluid height, may be calculated. Of course,care must be taken in order to ensure that acoustic radiation reflectedby the interface between the reservoir base and the fluid is discounted.

Thus, the present invention represents a significant improvement overknown technologies relating to the acoustic properties of the fluid in aplurality of reservoirs. As discussed above, the acoustic properties ofthe contents of fluid reservoirs typically involves placing a sensor indirect contact with the fluid. This means that the sensor must becleaned between each use to avoid cross-contamination of the fluid inthe reservoirs. In contrast, the invention allows for assessment of thefluid in a plurality of containers without direct contact with the fluidin the containers.

While other non-contact acoustic systems are known in the art, suchsystems provide only an indirect and approximate assessment of the fluidin a reservoir. For example, the acoustic system described in U.S. Pat.No. 5,880,364 to Dam employs a technique in which the acoustic radiationis transmitted from a sensor through an air-containing portion of thecontainer and then reflected from the air-liquid interface of thecontainer back to the sensor. The round trip transit time is used todetermine the volume of the air-containing portion of the container. Thevolume of liquid in the container is determined by subtracting thevolume of the container not occupied by the liquid from the volume ofthe entire container. One drawback, of this technique is that it cannotprovide an accurate assessment of the liquid volume in a container whenthe volume of the container is not precisely known. This is particularlyproblematic when small reservoirs such as those typically used incombinatorial techniques are employed. The dimensional variability forsuch containers is relatively large when, considered in view of thesmall volume of the reservoirs. Furthermore, the technique cannot beemployed when the volume of the container is completely unknown oralterable. Finally, since acoustic radiation never penetrates theliquid, the reflected radiation can at best only provide informationrelating to the surface of the liquid, not information relating to thebulk of the liquid. There would be little difference in the amplitude ofthe reflection from the liquid based on its composition since theacoustic impedance of air is essentially zero. Such a negligibleimpedance would cause essentially all the acoustic energy to bereflected from the fluid surface, so changes in the liquid impedancecould not be detected based on changes in the reflection. Such changeswould be lost in the accompanying noise.

In contrast, because the invention involves the transmission of acousticradiation through the portion of each reservoir adapted to contain afluid, the transmitted acoustic radiation may provide informationrelating to the volume as well as the properties of the fluid in thereservoir, such as acoustic impedance and the presence of impurities.For example, the invention provides a plurality of reservoirs, wherein aportion of each reservoir is adapted to contain a fluid. A fluidcontained in a reservoir must ordinarily contact a solid surface of thereservoir. When the invention is employed in a reflective mode, some ofthe generated acoustic radiation may be reflected by the interfacebetween the fluid and the solid surface, while the remainder istransmitted through the fluid contained in the reservoir. Thetransmitted radiation is then reflected by another surface, e.g., a freesurface, of the fluid contained in the reservoir. By determining thedifference in round-trip transit time between the two portions, thevolume of the fluid in the reservoir may be accurately determined. Inaddition, transmission of acoustic radiation through the fluid allowscharacteristics of the acoustic radiation to be altered by that fluid.Thus, information relating to a property of the fluid may be deduced byanalyzing a characteristic of the transmitted acoustic radiation.

In addition, air, like other gases, exhibits low acoustic impedance, andacoustic radiation fends to attenuate more in gaseous materials than inliquid or solid materials. For example, the attenuation at 1 MHZ for airis approximately 10 dB/cm while that of water is 0.002 dB/cm. Since theacoustic system described in U.S. Pat. No. 5,880,364 to Dam requiresacoustic radiation to travel through air, this system requires much moreenergy to operate. Thus, the present invention represents a more energyefficient technology that may be employed to provide more accurate anddetailed information about the properties of the fluid in each of aplurality of fluid reservoirs. Some of this additional accuracy can beachieved by using higher frequency acoustic waves (and hence shorterwavelengths), as these acoustic waves can be transmitted effectivelythrough fluids yet would be very rapidly attenuated in air.

It will be appreciated by those of ordinary skill in the art thatconventional or modified sonar techniques may be employed. Thus, theacoustic radiation will be reflected back at the piezoelectric element36, where the acoustic energy will be converted into electrical energyfor analysis. The analysis may be used, for example, to reveal whetherthe reservoir contains any fluid at all. If fluid is present in thereservoir, the location and the orientation of the free fluid surfacewithin the reservoir may be determined, as well as the overall volume ofthe fluid. Characteristics of the reflected acoustic radiation may beanalyzed in order to assess the spatial relationship between theacoustic radiation generator and the fluid surface, the spatialrelationship between a solid surface of the reservoir and the fluidsurface, as well as to determine a property of the fluid in eachreservoir, e.g., viscosity, surface tension, acoustic impedance,acoustic attenuation, solid content, and impurity content. Once theanalysis has been performed, a decision may be made as to whether and/orbow to dispense fluid from the reservoir.

Depending on the type of assessment to be carried out, varioustechniques known in the art may be adapted for use in the presentinvention. Generally, interfacial energy measurements are routinelycarried out using contact-angle measurement. The present invention maybe adapted to perform such contact-angle measurements. In addition, anumber of other acoustic assessment techniques are known in the art. Forexample, U.S. Pat. No. 4,391,129 to Trinh described a system formonitoring the physical characteristics of fluids. The physicalcharacteristic may be determined from acoustic assessment of theinterfacial tension of fluids to a high degree of accuracy. U.S. Pat.No. 4,558,589 to Hemmes describes an ultrasonic blood-coagulationmonitor. U.S. Pat. No. 5,056,357 to Dymling et al. described acousticmethods for measuring properties in fluids through Doppler shifts. Otheracoustic assessment techniques that may be adapted for use in thepresent invention are described, for example, in U.S. Pat. Nos.4,901,245; 5,255,564; 5,410,518; 5,471,872; 5,533,402; 5,594,165;5,623,095; 5,739,432; 5,767,407; 5,793,705; 5,864,698; 6,119,510;6,227,040; and 6,298,726.

In order to form a biomolecular array on a substrate using the inventiveapparatus, substrate 45 is positioned above and. in proximity to thefirst reservoir 13 such that one surface of the substrate, shown in FIG.1 as underside surface 51, faces the reservoir and is substantiallyparallel to the surface 17 of the fluid 14 therein. Once the ejector,the reservoir, and the substrate are in proper alignment, the acousticradiation generator 38 is activated to produce acoustic radiation thatis directed by the focusing means 37 to a focal point 47 near the fluidsurface 17 of the first reservoir. That is, an ejection acoustic wavehaving a focal, point near the fluid surface is generated in order toeject at least one droplet of the fluid, wherein the optimum intensityand directionality of the ejection acoustic wave is determined using theaforementioned analysis, optionally in combination with additional data.The “optimum” intensity and directionality are generally selected toproduce droplets of consistent size and velocity. For example, thedesired intensify and directionality of the ejection acoustic wave maybe determined by using the data from the above-described assessmentrelating to reservoir volume or fluid property data, as well asgeometric data associated with the reservoir. In addition, the data mayshow the need to adjust the relative position of the acoustic radiationgenerator with respect to the fluid surface, in order to ensure that thefocal point of the ejection, acoustic wave is near the fluid surface,where desired. For example, if analysis reveals that the acousticradiation generator is positioned such that the ejection acoustic wavecannot be focused near the fluid surface; the acoustic radiationgenerator and/or the reservoir is repositioned using vertical,horizontal, and/or rotational movement to allow appropriate focusing ofthe ejection acoustic wave.

As a result, droplet 49 is ejected from the fluid surface 17 onto adesignated site on the underside surface 51 of the substrate. Theejected droplet may be retained on the substrate surface by solidifyingthereon after contact; in such an embodiment, it may be necessary tomaintain the substrate at a low temperature, i.e., a temperature thatresults in droplet solidification after contact. Alternatively, or inaddition, a molecular moiety within the droplet attaches to thesubstrate surface after contract, through adsorption, physicalimmobilization, or covalent binding.

Then, as shown in FIG. 1B, a substrate positioning means 50 repositionsthe substrate 45 over reservoir 15 in order to receive a droplet therefrom at a second designated site. FIG. 1B also shows that the ejector 33has been repositioned by the ejector positioning means 43 belowreservoir 15 and in acoustically coupled relationship thereto by virtueof acoustic coupling medium 41. Once properly aligned, the acousticradiation generator 35 of ejector 33 is activated to produce low energyacoustic radiation to assess the fluid in the reservoir 15 and todetermine whether and/or how to eject fluid from the reservoir.Historical droplet election data associated with the ejection sequencemay be employed as well. Again, there may be a need to reposition, theejector and/or the reservoir so as to create the proper distance betweenthe acoustic radiation generator and the fluid surface, in order toensure that the fecal point of the ejection acoustic wave is near thefluid surface, where desired. Should the results of the assessmentindicate that fluid may be dispensed from the reservoir, focusing means37 is employed to direct higher energy acoustic radiation to a focalpoint 48 within fluid 16 near the fluid surface 19, thereby ejectingdroplet 53 onto the substrate 45.

It will be appreciated that various components of the apparatus mayrequire individual control or synchronization to form an array on asubstrate. For example, the ejector and/or reservoir positioning meansmay be adapted to eject droplets from each reservoir in a predeterminedsequence associated with an array to be prepared on a substrate surface.Similarly, the substrate positioning means for positioning the substratesurface with respect to the ejector and/or reservoir may be adapted toposition the substrate surface to receive droplets in a pattern or arraythereon. Any or all positioning means, i.e., the ejector positioningmeans, the reservoir positioning means and the substrate positioningmeans, may be constructed from, for example, motors, levers, pulleys,gears, a combination thereof, or other electromechanical or mechanicalmeans known to one of ordinary skill in the art. It is preferable toensure that there is a correspondence between the movement of thesubstrate, the movement of the reservoirs, the movement of the ejector,and the activation of the ejector to ensure proper array formation.

Accordingly, the invention relates to the assessment of the fluid in aplurality of reservoirs as well as to dispensing a plurality of fluidsfrom reservoirs, e.g., in order to form a pattern or an array, on thesubstrate surface 51. However, there are a number of different ways inwhich content assessment and fluid dispensing may relate to each other.That is, a number of different sequences may be employed for assessingthe fluid in the reservoirs and for dispensing fluids there from. Insome instances, the fluid in a plurality of reservoirs may be assessedbefore fluid is dispensed from any of the reservoirs. In otherinstances, the fluid in each reservoir may be assessed immediatelybefore fluid is dispensed there from. The sequence used typicallydepends on the particular fluid-dispensing technique employed as well asthe intended purpose of the sequence.

FIG. 2 illustrates an example of the inventive apparatus that providesfor assessment of the fluid in a plurality of reservoirs in transmissivemode rather than in reflective mode. Considerations for the design andconstruction of this apparatus are similar to those discussed above.Thus, an apparatus 11′ includes a first reservoir 13′ and a secondreservoir 15′, each adapted to contain a fluid indicated at 14′ and 16′,respectively, and each of substantially identical construction. Thefirst reservoir 13′is depicted in an open state, while the secondreservoir is depicted in a sealed state. An acoustic radiation generator35′ is positioned below the reservoirs, and analyzer 38′ is positionedin opposing relationship with the acoustic radiation generator 35′ abovethe reservoirs.

In operation, the fluid in each of the reservoirs is acousticallyevaluated before pipette 60′ is employed to dispense fund there from. Asshown, the contents 14′ of the first reservoir 13′ have already beersacoustically assessed. As the assessment has revealed that the firstreservoir 13′ contains at least a minimum acceptable level of fluid 14′,the first reservoir 13′ is open and ready for fund to be dispensed therefrom via pipette 60′. The consents 16′ of the second reservoir 15′ areundergoing acoustic assessment, us depicted by FIG. 2, as the secondreservoir 15′ is interposed between the acoustic radiation-generator 35′and the analyzer 38′. The acoustic radiation generator 35′ and theanalyzer 38′ are acoustically coupled so she second reservoir viacoupling media 41′ and 42′respectively. Once the acoustic radiationgenerator 35′, the second reservoir 15′, and the analyzer 38′ are inproper alignment, the acoustic radiation generator 35′ is activated toproduce acoustic radiation that is transmitted through the reservoir 15′and its contents 16′ toward the analyzer 38′. The received acousticradiation is analyzed by an analyzer 38′ as described above

FIG. 3 schematically illustrates an exemplary rectilinear array ofreservoirs in a well plate that may be used with the invention. Wellplate configurations can include from 96, 192, 384, to as many as100,000 wells on a plate. Such configurations may also be asymmetric.The configuration of FIG. 3 is provided for simplicity of understanding.The reservoir array is provided in the form of a well plate 61 havingthree rows and two columns of wells. As depicted in FIGS. 3A and 3C,wells of the first, second, and third rows of wells are indicated at 63Aand 63B, 65A and 65B, and 67A and 67B, respectively. Each is adapted tocontain a fluid having a fluid surface. As depicted in FIG. 3B, forexample, reservoirs 63A, 65A, and 67A contain fluids 64A, 66A, and 68A,respectively. The fluid surfaces for each fluid are indicated at 64AS,66AS, and 68AS. As shown, the reservoirs have a height-to diameter-ratioless than one and are of substantially identical construction so as tobe substantially acoustically indistinguishable, but identicalconstruction is not a requirement. Each of the depicted reservoirs isaxially symmetric, having vertical walls extending upward from circularreservoir bases indicated at 63AB, 63BB, 65AB, 65BB, 67AB, and 67BB, andterminating at corresponding openings indicated at 63AO, 63BO, 65AO,65BO, 67AO, and 67BO. The bases of the reservoirs form a common exteriorlower surface 69 that is substantially planar. Although a full wellplate skirt (not shown) may be employed that extends from all edges ofthe lower well plate surface, as depicted, partial well plate skirt 71extends downwardly from the longer opposing edges of the lower surface69. The material and thickness of the reservoir bases are such thatacoustic radiation may be transmitted there through and into the fluidcontained within the reservoirs.

Measurements were performed to determine the utility of echoing off afluid surface in a reservoir at a sub threshold level, as a meaningfulway to set ejection power. A perturbation pulse of 100 μsec duration wasused to generate a surface perturbation or dimple. Then a series of 50perturbation interrogation pulses are excited by a tone burst having thesame wave form as the initial waveform, every 50 μsec after the initialpulse. The center frequency of the tone burst was 10 MHz.

From the data, echo “images” were constructed. In FIGS. 4A-4C, along thehorizontal axis, each column in the image corresponds to a perturbationinterrogation pulse response signal displayed using a gray-scaleintensity corresponding to its relative amplitude. The firstperturbation interrogation pulse is launched 200 μsec after the initialtone burst excitation. It should be noted that the duration of the pulseis selected to approximate that of a drop forming pulse. In general,smaller drops require shorter durations and could be 0.5 μsec orshorter. Larger drops may require durations of 1000 μsec or longer.

The response from this perturbation interrogation pulse is plotted inthe leftmost column in the image. The next column in the image is theperturbation interrogation pulse response, from the second perturbationinterrogation pulse, which launched 50 μsec after the first perturbationinterrogation pulse, and so on. Along the vertical direction of theimage, ranging from the top down, the first 2000 nanoseconds of eachperturbation interrogation pulse response are quantized into pixels andcomposited to form a grayscale image.

The echo “images” pertain to different DMSO concentrations. In FIGS.4A-4C are shown echo images of 70% DMSO and 90% DMSO, for tone burstexcitations of 1 dB (FIG. 4A), 0.5 dB (FIG. 4B), and 0 dB (FIG. 4C)below an ejection threshold level.

From the figures it is evident that the images are remarkably similaracross the two DMSO concentrations. The tone burst excitation levels forthe different DMSO concentrations are always quoted relative to theejection threshold for those concentrations, but the absolute ejectionthresholds are quite different between 70% and 90% DMSO (on the order of1.0 dB). This corresponds mostly to the significant difference inviscosity between 70% and 90% DMSO.

Thus, even though the absolute power set points are quite differentbetween each pair of images, the images appear very similar. This“universality” of the echo images, when considered relative to ejectionthreshold, implies that it should be possible to use the echo images asa means to determine the ejection threshold power, without needing toknow the DMSO concentration. (Presumably, one could determine the DMSOconcentration using echoing, by looking at the fluid surfaceperturbation over longer time scales, and hence extracting the viscosityof the fluid. But such a measurement could be somewhat slow for someapplications). Refer to U.S. patent application Ser. No. 10/310,638,entitled “Acoustic Assessment of Fluids in a Plurality of Reservoirs,”assigned to the assignee of the subject application and hereinincorporated by reference for a more detailed discussion of methods formeasuring DMSO content of a fluid in a reservoir as well means formeasuring such fluid properties as, viscosity and surface tension.

Using images like those shown in FIGS. 4A-4C, one can determine thedroplet ejection threshold level of an unknown fluid by extractingsimilar features from one or two echo images made using perturbationpulses or tone bursts, to capture a value that corresponds to the “gap”between two echoes from a given pulse as they split (along the verticaldirection) in the range between 0 to 15 along the horizontal axis of theimages. This gap, which presumably corresponds to size of the fluiddimple caused by the perturbation of the fluid surface from the acousticenergy and can be used to quantify information in the images.

FIG. 5 shows a chart of the maximum “gap” between the split echoes, forthe tone burst powers and DMSO concentrations associated with FIGS. 4Athrough 4C. While the curve of FIG. 5 provides an alternative means topredict droplet ejection threshold energy, from an echo image associatedwith a particular perturbation tone burst, the method is laborintensive, with significantly increased processing times.

For example, if a tone burst of energy 0.71 dB below droplet ejectionthreshold was used, and the resulting echo image was processed to obtaina maximum “gap” between the split echoes of 2536 ns, when the quadraticcurve of FIG. 5 was inverted, the resulting curve corresponds to a toneburst power 0.68 dB below threshold. Thus, from the echo image usedhere, it is possible to predict the ejection threshold energy to within0.03 dB.

Using a data processing method employing the Fast Fourier Transform(FFT), a single echo can be collected and processed to provide similarestimations of the required additional acoustic energy to create adroplet at the threshold of ejection. This has substantial advantagesover the process used supra to product the better feature extractionalgorithms for the images shown in FIGS. 4A-4C. Processing echomeasurements using the FFT approach substantially improves theefficiency of fluid delivery for many drop ejection applications. Goingfrom the time domain to the frequency domain is the key feature inenabling one to use an echo pulse (rather than many measuredreflections) to get a good prediction of how much more energy isrequired to get to threshold. While the FFT approach to data analysisoperates in the frequency domain and may be more efficient, this is justone approach to the analysis of acoustic data, other approaches arepossible and useful. For example, a method of data analysis in the timedomain is set forth infra.

A method useful in the analysis of acoustic “echo” data is shown in FIG.6. To determine the amount of acoustic energy necessary to increase aperturbation poise to the level of a droplet forming pulse, thealgorithm 200 includes the following steps.

In an initial optional first step 201A, a ranging pulse can be used todetermine the fluid height in the well so that the coupling of thetransducer and the fluid surface is sufficient for the perturbationpulse to be in focus at the site. This information also provides data onthe range in time where acoustic energy reflected from the fluid surfacewould be expected to return to the transducer, and this can reduces theamount of data sampling and expedites analysis of the pulse echoes

In an initial optional second step 201B, a background noise pulse can beused to determine the magnitude of the background noise from acousticreflections from the fluid surface. This background noise result canthen be applied to extract background noise from other reflectedacoustic pulses from the fluid surface. For example, knowledge of thebackground noise characteristics of reflected signals can be used toimprove processing of signals (i.e., the signal to noise ratio) of theperturbation interrogation pulse echo and such techniques are familiarto those of skill in the art.

Optionally, the ranging pulse and the background noise pulse can be thesame pulse 201. The reflected energy in the “combined” pulse can beprocessed to extract both the ranging information and the backgroundnoise characteristics.

In a next step 203, a perturbation pulse is sent to the fluid site toperturb the fluid surface and preferably is not a drop forming pulse,but a pulse with a similar waveform at a lower energy level.

In a next step 205, one or more perturbation interrogation pulses aresent to the fluid site to be reflected from the now perturbed fluidsurface.

Optionally, the pulses of steps 203 and 205 can be combined into asingle waveform 207.

In step 209, the reflected acoustic energy from the one or moreperturbation interrogation pulses of step 205 form the perturbationinterrogation pulse echo(s) and are collected by the transducer foranalysis.

In step 211, the data is processed to estimate the gap between the powerin the perturbation pulse and the power required for a drop formingpulse. This step can be accomplished by a variety of means as describedelsewhere in more detail, and it can optionally include the results ofthe analysis of the ranging pulse, background noise pulse in addition tothe one or more perturbation interrogation pulses. The analysis canconsist of either processing in the time or frequency domain.

In step 213, the result of the gap in drop forming power is recorded.This data and other associated information can be stored in a data baseor other comparable format for later use in plate auditing. The datacollection means can be any suitable media provided with the analyzer,such as tape, CD-ROM or other media formats.

An example of algorithm 200, assumes the ranging pulse and thebackground noise pulse information is not required. A perturbation pulseis about 275 microseconds in length is sent to the fluid site. After aninterval of about 300 microseconds a single perturbation interrogationpulse are sent as a combined waveform about 575 microseconds long. Thereflected pulse or the perturbation interrogation pulse response issimilar in length (about 600 microseconds). The portion of theperturbation interrogation pulse response that is subject to processingby an FFT to determine spacing between two minima (the perturbationinterrogation pulse “echo”) is only about 2 microsecond long and occursabout 550 to 600 microseconds into the perturbation interrogation pulseresponse. The selection of the time domain data for FFT processing isbased on the ranging pulse echo time, background noise data and thelocation of the low frequency preamble.

FIG. 7 a is a time trace, essentially equivalent to one column in FIG.4. The spacing between minima in FIG. 7 c is inversely related to the‘gap’ between the split echoes of FIG 4. While using the FFT is moreefficient as an algorithm, in theory one could measure the gap along agiven column of FIG. 4 to obtain comparable information to create thechart shown. It is possible to map one minima spacing in the FFT to aparticular energy below ejection threshold, only because there is amodel for relating the spacing to the threshold energy i.e., energyrelative to ejection threshold=A*in(spacing)+B. This relationship holdswell for different fluids, different acoustic frequencies and otherparameters. The A and B parameters are determined off-line before makingthe measurement on a given plate. The A and B parameters will varysomewhat with acoustic frequency, fluid composition and relatedparameters. A and B are more sensitive to frequency than DMSOconcentration.

If the ‘gap’ between the split echoes was found in the time domain,there would be a relationship similar to FIG. 7, e.g., energy relativeto ejection threshold=A*ln(1/gap)+B. The flow chart of FIG. 6 is justthe best approach. With the use of advanced data processing techniques,perturbation interrogation pulse echoes can be processed to yieldcomparable data at a speed limited only by the processing power of theanalyzer used.

The perturbation interrogation pulse response is the entire acquiredsignal that comes back as the -perturbation interrogation pulse isreflected from the fluid surface responding to the earlier perturbationpulse. From this signal, the portion of the signal that contains theecho data from the perturbation interrogation pulse that is nominally200-300 μs past the perturbation tone burst. We subsequently proceed tooperate on the FFT of this time-domain data.

The Low-Frequency Preamble is the low frequency noise or interferencethat can interfere with the reflected signal and may have to be removedbefore the can be analyzed.

There are two other possible applications for the algorithm of FIG.6—one can envision these applications as iteratively invoking the seriesof steps 201-213 as shown in FIG. 6. One such application is thecreation of a plate “energy signature” by moving from well to well usingthe algorithm of FIG. 6 (once, or perhaps multiple times to average outany noise) and map the returned minima-spacing in MHz to energy belowdrop ejection threshold in dB. Holding everything constant (e.g., themagnitude of the perturbation pulse) and all things being equal (e.g.,the volume and composition of the fluid in each reservoir), as onetraverses across the plate, the variation in the energy below dropejection threshold provides a good measure as to the variation in energy(as a function of well position) that one needs to compensate for duringdrop ejection. This variation is attributed to the differences in thetransmission characteristics of the plate to acoustic energy. A map ofthe difference in energy from well to well can be saved for future usein determining the level of drop forming pulses for the wells of platesbelieved to have similar energy signatures. This mapping method does notrequire any new measurement algorithms as the measurement of dropejection threshold for each reservoir in the plate is the same as theabove application for finding the proper energy level for the dropforming pulse.

The second application is the use of the drop ejection threshold tocalibrate the power system of the acoustic radiation generator. Thisapproach is based on using a known volume of a fluid of knowncomposition in a well of known structure and composition. Previoustesting, as perhaps done in an audit or a controlled loading of areservoir, would provide a known threshold level for droplet ejection,for example, 8.0 dB. If an ejector was placed in acoustic couplingrelationship with the well and the power input to the ejector wasincreased until the threshold level was reached, it might be presumedthat the power input level of the ejector was at 8.0 dB. However if thepower level was decreased by a known increment, and the algorithm ofFIG. was performed at what was presumed to be a known lower level, andthe gap in power measured between the perturbation pulse and thethreshold of ejection plus this sub-threshold power setting did notequal 8.0 dB, the power setting for the ejector would be erroneous.However, a series of such incremental sub threshold measurements, withthe use of the algorithm like the algorithm of FIG. 6 would enable oneto do a power calibration of an acoustic ejector system. Thiscalibration would bring the expected power required for ejection in linewith the actual power for ejection. Note that since the response of theacoustic power system changes with frequency content of the pulses, thiscalibration would best be performed for pulses with frequency contentthat matched the frequency content required for the drop forming pulseof the said known fluid composition. Hence, if multiple fluidcompositions were present on a single substrate, optimally, the powersystem calibration would perform for each fluid composition using theappropriate wave form associated with the drop forming pulse for eachfluid composition.

One approach to the calibration system is set forth below. In theexample provided two processes are linked together, both of which arebased on the algorithm of FIG. 6: in (a) the amplitude of theperturbation interrogation pulse is incrementally increased until thedrop ejection threshold is reached; and in (b) a z-sweep is performed,i.e., the process of iteratively translating in transducer height, or zposition, in equal increments, and invoking the algorithm of FIG. 6 ateach position. The entire process is set forth in steps 1-3 below.

1. Incrementally increase the amplitude of the perturbationinterrogation pulse, and run the algorithm of FIG. 6 at each amplitudesetting. Convert minima-spacing to energy below drop ejection thresholdfor each amplitude setting. As soon as the drop ejection threshold (i.e.the energy below drop ejection threshold is about 0 dB) is reached,perform a linear regression and set the amplitude of the perturbationinterrogation pulse to the calculated drop ejection threshold.

2. Determine the optimal transducer z position at this amplitudesetting, by performing a z-sweep and subsequently fitting & quadraticfunction to the resulting transducer z position versus a function basedon the spacing between two minima, i.e., y=f(z). The minima of thequadratic function yield the z-position at which the minimum spacingbetween two minima at the power setting of (1) occurs. This transducerz-position is the optimal position at the power setting found in (1) toeject a droplet.

3. Perform (1) again, at the optimal z-position found in (2), to furtherrefine the precise amplitude setting of the perturbation interrogationpulse.

Once both the optimal transducer z-position and power setting have beenmeasured, by proceeding through steps 1-3, generate a small number ofperturbation interrogation pulses, at the power setting found in (3) andz position found in (2), measuring the energy delivered to thetransducer. This energy level (measured in the system of the presentinvention in mJ) is then used to calibrate the power system of theapparatus.

It should be evident, then, that the invention provides a number ofpreviously unrealized advantages for assessing the fluid in a pluralityof reservoirs. First, acoustic assessment is a generally noninvasivetechnique that may be carried out regardless of whether the reservoirsare sealed or open. That is, acoustic assessment does not requireextracting a sample for analysis or other mechanical contact that mayresult in sample cross-contamination. In addition, unlike opticaldetection techniques, optically translucent or transparent reservoirsare not required. This, of course, provides a wider range of choices formaterial that may be employed for reservoir construction. In addition,the use of opaque material would be particularly advantageous ininstances wherein the reservoirs are constructed to containphotosensitive fluids.

Thus, variations of the present invention will be apparent to those ofordinary skill in the art. For example, while FIG. 1 depicts theinventive apparatus in operation to form a biomolecular array bound to asubstrate, the apparatus may be operated in a similar manner to format aplurality of fluids, e.g., to transfer fluids from odd-sized bulkcontainers to wells of a standardized well plate. Similarly, while FIG.2 illustrates that the acoustic radiation generator and the detector arein vertical opposing relationship, other spatial and/or geometricarrangements may be employed so long as acoustic radiation generated istransmitted through at least a portion of the reservoir to the detector.

In addition, the invention may be constructed as to be highly compatiblewith existing infrastructure of materials discovery and with existingautomation systems for materials handling. For example, the inventionmay be adapted for use as an alternative or a supplement to contentassessment means that are based on optical detection. In some instances,sonic markers may be provided in the reservoirs to identify the fluid inthe reservoir. Thus, the invention may be employed as a means forinventory identification and control in a number of contexts, including,but not limited, to biological, biochemical, and chemical discovery andanalysis. Also, differences in the acoustic transmission behaviors ofdifferent, reservoirs and their contents can be measured by the presentinvention and used to modify the drop forming pulse to improveconsistency in energy levels and droplet volumes for droplet ejection.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention. Other aspects, advantages, and modifications will beapparent to those skilled in the art to which the invention pertains.

All patents, patent applications, journal articles, and other referencescited herein are incorporated by reference in their entireties.

1.-53. (canceled)
 54. A method for characterizing a plurality of fluidreservoirs contained in or located on a substrate by (a) providing adeterminable volume fluid having a known composition in each reservoirand adjusting the fluid volume in each reservoir to conform thereservoir to a predetermined ejection threshold level, (b) acousticallycoupling a first reservoir to an acoustic ejector that produces acousticradiation, (c) activating the ejector to generate a sub-threshold pulseof acoustic radiation through the substrate and into the fluid, whereinthe sub-threshold pulse's acoustic energy lies below the level effectiveto eject a droplet from the first reservoir, (d) analyzing the effect ofthe sub-threshold pulse to determine the gap to the ejection threshold,(e) comparing the ejection threshold with the predetermined ejectionthreshold, (f) repeating (a), (b), (c), (d) and (e) with each of theplurality of fluid reservoirs in succession, and: (g) using thedifference between ejection threshold and predetermined ejectionthreshold at each site to characterize reservoir-to-reservoir variationson the substrate.
 55. A method for analysis of acoustic data todetermine the amount of acoustic energy necessary to increase aperturbation pulse to the level of a droplet ejection pulse, the methodcomprising the following steps: (a) delivering a perturbation pulse tothe unperturbed surface of a fluid in a well to determine (1) fluidheight and/or (2) the magnitude of the background noise sensed by anacoustic detector; (b) delivering a perturbation interrogation pulse tothe fluid surface; (c) processing the reflected acoustic energy from theperturbation interrogation pulse; (d) removing low frequency noise fromthe signal processed in step (c) and reprocessing that signal ifnecessary; (e) determining all of the minima in the frequency spectrumof the signal output in step (d); and (f) selecting the two minima ofinterest, based on the frequency content of the processed signal, anddetermining the spacing between the two minima.
 56. The method of claim55, wherein step (c) includes using fluid height to determine where tolook for an echo portion of the reflected acoustic energy from theperturbation interrogation pulse and where low frequency noise resides.57. The method of claim 55, wherein step (c) includes using themagnitude of the background noise to extract an echo portion of thereflected acoustic energy from the perturbation interrogation pulse. 58.The method of claim 57, wherein the step of extracting an echo portionis performed by an analyzer.
 59. The method of claim 58, wherein theprocessing in step (c) includes computing a Fast Fourier Transform ofthe extracted echo portion.
 60. The method of claim 59, wherein theanalyzer looks for the presence of a low frequency preamble and removesit from the time-domain signal if found, and then recomputes the FastFourier Transform.
 61. A method for analysis of acoustic data todetermine the amount of acoustic energy necessary to increase a pulse toan acoustic energy level sufficient for droplet ejection, the methodcomprising the steps of: (a) delivering a perturbation pulse to theunperturbed surface of a fluid in a well to determine (1) fluid heightand (2) the magnitude of the background noise; (b) delivering aperturbation interrogation pulse to the fluid surface; (c) using ananalyzer to compute the FFT of the reflection of the perturbationinterrogation pulse from the fluid surface; (d) using the analyzer tolook for the presence of a low frequency preamble in the reflection ofthe perturbation interrogation pulse and, if a low frequency preamble isfound, to remove it from the reflection and to compute the FFT of thereflection with the low frequency preamble removed; (e) determining allof the minima in the FFT computed in steps (c) or (d); and (f) selectingtwo minima of interest, taking as in input the frequency content of theperturbation pulse, and determining the spacing between the two minimaselected.
 62. The method of claim 61 wherein an acoustic generator isacoustically coupled to a plurality of wells in a well plate, whereinthe spacing between two minima is computed for each well plate to whichthe acoustic generator is coupled, and wherein the spacing between twominima is converted for each well plate to which the acoustic generatoris coupled into an energy below threshold value, thereby providing ameasure of the variation in energy as a function of well position neededto compensate for well-to-well variation during droplet ejection andcreating a plate energy signature.
 63. The method of claim 62 whereinthe spacing between two minima is determined multiple times for at leastone well of the well plate, thereby providing a number of values of thespacing which can be averaged to reduce noise.
 64. The method of claim62 wherein the perturbation pulse delivered in step (a) hassubstantially the same energy for each of the well plates for which thespacing between two minima is computed.
 65. A method for the analysis ofacoustic data to determine the amount of additional acoustic energynecessary to change a pulse to the level of a droplet-forming pulse fora fluid surface in a well containing a fluid, the method comprising thesteps of: (a) using a ranging pulse to determine the fluid height in thewell; (b) sending a perturbation pulse to a fluid site near the fluidsurface to perturb the fluid surface; (c) sending at least one batherpulse to the fluid site to be reflected from the perturbed fluidsurface; (d) using the reflected acoustic energy from the at least onefurther pulse of step (c) to determine a waveform for analysis; and (e)using an analyzer to process the waveform to estimate the gap betweenthe power in the perturbation pulse and foe power required for adroplet-forming pulse.
 66. The method of claim 65, further comprisingthe step (a′) of using a background noise pulse to determine themagnitude of the background noise from acoustic reflections from thefluid surface.
 67. The method of claim 66, further comprising the stepof using the reflection from the background noise pulse to removebackground noise from other reflected acoustic pulses from the fluidsurface, thereby improving processing of the reflection from the furtherpulse.
 68. The method of claim 66, wherein the ranging pulse and thebackground noise pulse are the same pulse.
 69. The method of claim 65wherein the processing of step (e) is performed in the time domain. 70.The method of claim 65 wherein the processing of step (c) is performedin the frequency domain.
 71. The method of claim 65 wherein the gapestimated in step (e) is recorded in a data collection format.
 72. Themethod of claim 71 wherein the data collection format is a database. 73.The method of claim 65 wherein steps (a)-(e) are iteratively invoked tocreate a plate energy signature by determining the gap of step (e) foreach of a plurality of wells in a well plate.
 74. The method of claim 73wherein the magnitude of the perturbation pulse is substantially thesame for each well for which steps (a)-(e) are carried out, the volumeand composition of the fluid in each well is substantially the same. 75.The method of claim 73 further comprising the step of saving the plateenergy signature for further use.
 76. The method of claim 65 whereinsteps (a-e) are performed repeatedly with perturbation pulses ofincreasing energy below the droplet ejection threshold, and areperformed repeatedly for different positions of a transducer whichgenerates the pulses.
 77. An apparatus for acoustically ejecting adroplet of fluid from a reservoir, the apparatus comprising: an acousticradiation generator coupled to a reservoir and configured to: generate aperturbation pulse of acoustic radiation to disturb a surface of a fluiddisposed in the reservoir, the perturbation pulse being associated witha perturbation acoustic energy not sufficient to eject any droplet ofthe fluid from the reservoir; and generate, after the perturbationpulse, a perturbation interrogation pulse of acoustic energy such thatthe perturbation interrogation pulse is at least partially reflected bythe disturbed surface of the fluid to form a reflected interrogationpulse response; and an analyzer configured to: detect the reflectedinterrogation pulse response; process information associated with thedetected reflected interrogation pulse response; and determine adifference between the perturbation acoustic energy and an ejectionacoustic energy based on at least information associated with thedetected reflected interrogation pulse response, the ejection acousticenergy being sufficient to eject a droplet of the fluid from thereservoir.
 78. The apparatus of claim 77 wherein the acoustic radiationgenerator is further configured to, before the perturbation pulse andthe perturbation interrogation pulse, generate a ranging interrogationpulse in order to determine a height of the surface of the fluiddisposed in the reservoir.
 79. The apparatus of claim 78 wherein theacoustic radiation generator is further configured to, after the ranginginterrogation pulse but before the perturbation pulse and theperturbation interrogation pulse, adjust a position of the acousticradiation generator with respect to the reservoir based on at leastinformation associated with the height of the surface of the fluiddisposed in the reservoir so that the perturbation pulse is generatedand focused at a site at or near the surface of the fluid.
 80. Theapparatus of claim 77 wherein the acoustic radiation generator isfurther configured to, after the perturbation pulse and the perturbationinterrogation pulse, generate an ejection pulse of acoustic radiationassociated with the ejection acoustic energy in a manner effective toeject a droplet of the fluid from the reservoir.
 81. The apparatus ofclaim 77 wherein the acoustic radiation generator is coupled to thereservoir through at least a focusing component and an acoustic couplingmedium.
 82. The apparatus of claim 77 wherein the acoustic radiationgenerator includes a transducer.
 83. The apparatus of claim 82 whereinthe analyzer includes the transducer.
 84. The apparatus of claim 77wherein the analyzer is further configured to analyze a frequencyspectrum of a portion of the detected reflected interrogation pulseresponse.
 85. The apparatus of claim 84 wherein the portion of thedetected reflected interrogation pulse response is less than 1% of thedetected reflected interrogation pulse response.
 86. The apparatus ofclaim 84 wherein the analyzer is further configured to use a frequencydomain-based algorithm to identify a difference in frequency between twominima of the frequency spectrum of the portion of the detectedreflected interrogation pulse response.
 87. The apparatus of claim 86wherein the frequency domain-based algorithm is a Fast FourierTransform-based algorithm.